Engineering lower inhibitor affinities in β- d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.

Jordan, Douglas; Wagschal, Kurt; Fan, Zhanmin; Yuan, Ling; Braker, Jay; Heng, Chamroeun

β- d-Xylosidase/α- l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-β- d-xylooligosaccharides to d-xylose. One property that could use improvement is its relatively high affinities for d-glucose and d-xylose ( K ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K and K twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K values that are 1.5-2.7-fold ( d-glucose) and 1.9-4.6-fold ( d-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k than that of the wild type. At 0.1 M d-xylobiose and 0.1 M d-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.

ENZYME inhibitors; MICROBIAL enzymes; SITE-specific mutagenesis; GLYCOSIDASES; BIOMASS energy; LIGNOCELLULOSE; HYDROLASES

Journal of Industrial Microbiology & Biotechnology, 2011, Vol 38, Issue 11, p1821

1367-5435

Academic Journal

10.1007/s10295-011-0971-2